The present invention relates Hepatitis-C virus (HCV), specifically to expression and purification of an RNA-dependent RNA polymerase (RDRP) encoded by the HCV genome, to antibodies directed against HCV-RDRP and to methods of using the enzyme to diagnose chronic HCV infections and to screen for antiviral agents effective against HCV.
HCV is the major causative agent for post-transfusion and for sporadic non A, non B hepatitis (Alter, H. J. (1990) J. Gastro. Hepatol. 1:78-94; Dienstag, J. L. (1983) Gastro 85:439-462). Despite improved screening, HCV still accounts for at least 25 % of the acute viral hepatitis in many countries (Alter, H. J. (1990) supra; Dienstag, J. L. (1983) supra; Alter, M. J. et al. (1990a) J.A.M.A. 264:2231-2235; Alter, M. J. et al (1992) N. Engl J. Med. 327:1899-1905; Alter, M. J. et al .(1990b) N. Engl J. Med. 321:1494-1500). Infection by HCV is insidious in a high proportion of chronically infected (and infectious) carriers who may not experience clinical symptoms for many years. The high rate of progression of acute infection to chronic infection (70-100%) and liver disease ( greater than 50%), its world-wide distribution and lack of a vaccine make HCV a significant cause of morbidity and mortality.
HCV is an enveloped virus whose genome is a 9.5 kb single-stranded RNA (sense(+)) encoding a single polyprotein that is processed by proteolysis to yield at least 9 proteins. HCV is related to pestiviruses and flaviviruses (Choo, Q-L. et al. (1989) Science 244:362-364; Choo, Q-L. et al. (1991) Proc. Natl. Acad. Sci. USA 88:2451-2455. Reinfection of previously HCV-infected chimpanzees suggests that protective immunity is transient or non-existent (Farci, P. et al (1992) Science 258:135-140). Furthermore, results of recent vaccine trials suggest that development of an effective vaccine is remote (Houghton, M. et al. (1994) 2nd Internat. Meeting on Hepatitis C (San Diego)). Attempted treatment of chronic HCV infection using existing antiviral agents produces low cure rates and serious side effects. (Dienstag, J. L. (1983) supra.)
The nucleotide sequence of the HCV genome has been cloned and a single open reading frame has been identified. Using a vaccinia virus expression system, several cleavage products have been tentatively identified. (Lin, C. et al. (1994) J. Virol. 68:5063-5073; Grakoui, A. et al. (1993) J. Virol. 67:1385-1395.) The various putative cleavage products were recognized by antibodies raised against various peptides synthesized from amino acid sequences deduced from various segments of the coding regions. Sizes of antibody-reactive peptides were estimated by SDS-PAGE (See FIG. 1). The non-structural protein designated 5B (NS5B) has been shown to have an amino-terminal sequence SMSY (Ser-Met-Ser-Tyr). The NS5B region encodes a 68 kd protein (p68) which contains an internal GDD (Gly-Asp-Asp) motif found in RNA-dependent RNA polymerases of other RNA viruses (Koonin, E. V. (1991) J. Gen. Virol. 72:2197-2206). However, no polymerase activity has been detected for HCV p68. In fact, the question has been raised that the 5B protein (p68) alone does not encode an active RNA-dependent RNA polymerase enzyme and that another subunit, possibly the NS5A gene product, is essential to catalytic activity. Prior attempts by the inventors and others to express the NS5B coding region as a fusion protein, using existing expression systems that facilitate purification of the fusion product and specific cleavage have failed to yield any active polymerase.
HCV, in common with other RNA viruses that employ direct RNA-RNA replication, has a high mutation rate. Independent isolates of HCV RNA have numerous sequence differences. Hagedorn, et al., (2000) Curr. Top. Microbiol. Immunol. 242:225-260, reviewed sequence variation in the NS5B sequence of 48 independent isolates. While it was possible to identify regions of conserved sequence, the interpretation of the data is difficult because only a few were known to encode an active RDRP. Even fewer were known to be a sequence of an infectious virus.
At the present time, infectivity of a given HCV strain can only be demonstrated in tests in chimpanzees, which severely limits the number of strains which can be tested. The number of RDRP sequences which have been tested for activity is limited, as described herein, by the necessity of modifying the N-terminus of the NS5B sequence to permit independent expression of RDRP in a recombinant host cell. Subsequent to the original priority hereof, others have isolated and expressed active RDRP. Lohmann, V., et al (1997) J. Virol. 71:8416-8428 GenBank Z97730, reported an active clone of HCV type 1b. The enzyme was shown to be active after deletion of either 25 or 55 amino acids of the C-terminus. Addition of an oligo-his tag permitted purification by nickel affinity chromatography of enzyme expressed in insect cells. Yamashita, T., et al (1998) J. Biol. Chem. 273:15479-15486 prepared an active RDRP from a clone of type 1b-JK1 (GenBank X65196). The authors thereof prepared a C-terminally deleted (xcex9421) RDRP fusion protein with glutathione-S transferase (GST) attached at the N-terminus. The enzyme was expressed in E. coli, yielding active enzyme with or without the GST tag, which served as an affinity purification ligand. The authors also reported three single amino acid replacements which abolished activity. Ferrari, E., et al (1999) reported several C-terminal deletion constructs xcex9416,19 21, 55 and 63 combined with oligo-his tags at either the N- or C-terminus. The enzymes were expressed in E. coli cells and purified with a chelated nickel column. RDRP enzymes of two HCV strains were studied, a 1a xe2x80x9cH77xe2x80x9d or xe2x80x9cHutchinsonxe2x80x9d isolate and a 1b xe2x80x9cBKxe2x80x9d isolate, although the RN sequences of the strains were not specifically identified. The oligo-his tags did not destroy activity, however enzymes having a C-terminal oligo-his tag had greater activity in an assay using a poly(C) homopolymer template. Luo, G.et al,(2000) J. Virol. 74:851-863 reported isolation of several NS5B clones from serum of an infected patient. Nearly half of the isolates had little or no RDRP activity. One isolate, which had the highest in vitro activity, was found to have a stop codon resulting in a deletion of 18 C-terminal amino acids. Mn++ was found to stimulate activity 20-fold compared to the activity in the presence of Mg++. No nucleotide or amino acid sequences were reported. Patent publication No. WO 99/29843 disclosed an isolated NS5B sequence and encoded RDRP, both full length and having a 21 amino acid deletion at the C-terminus. The source of the HCV was not given although the sequences appear to be related to type 1a. No data regarding activity of the encoded RDRP was disclosed. For a recent review, see Hagedorn, C. et al. (2000).
The present invention provides methods for making modified structures of the HCV-RDRP. The need to make modifications is due to the quasi-species nature of the virus, the fact that the protein appears intracellularly as the product of post-translational cleavage of a polyprotein, and in vitro insolubility of the isolated enzyme. The modifications described herein enable translation of the NS5B region of HCV RNA in transformed host cells, without the necessity of translating other virus-coded proteins at the same time. The second category relates to modifications at the C-terminus that contribute to solubility of the enzyme in aqueous media. The third category relates to individual amino acid substitutions which can be introduced to individual isolates encoding HCV-RDRP to enhance enzyme properties. As a consequence of the high mutation rate that occurs during HCV replication, individual isolates encode RDRP variants that vary in primary sequence and in functional attributes. These include, for example, reaction rate, template specificity, processivity, ease of purification, stability during purification and during storage and the like. Other advantages of the modifications will be apparent to those of ordinary skill in the art from the description herein.
The present invention provides a recombinant protein of HCV having RDRP activity (r-HCV-RDRP) obtainable by expression in a host eukaryote or prokaryote cell of a modified NS5B coding region of HCV. The modification includes addition at the amino terminus of a methionine residue and optionally from 1-20 additional amino acids interposed between the N-terminal methionine and the N-terminal serine of unmodified NS5B gene product. The modification also includes deletion at the amino terminus of up to 9 amino acids to provide an amino-terminal methionine. Two methionines occur naturally according to the deduced sequence of wild-type HCV-RDRP. Therefore, modification includes deletion to remove amino acids lying N-terminal to either methionine or, alternatively, deletion to some intermediate point between the two methionines plus addition of an N-terminal methionine codon. Other optional modifications include deletion of from 18 to 60 C-terminal amino acids and various amino acid substitutions throughout the protein, as described in detail herein. Deletion at the C-terminus improves solubility of isolated NS5B protein, without destroying activity, in in vitro assays. Individual amino acid substitutions in the protein can enhance enzyme specific activity, stability during purification, template specificity and other properties as described herein. A combination of deletions and insertions, within the limits described is also contemplated. Added amino acid sequence can be devised to create a specific protease cleavage site to permit post translational modification of the recombinant HCV-RDRP expression produce, in vivo or in vitro. Such post-transcriptional modification can be used to generate exactly the amino acid sequence encoded by NS5B, having an N-terminal serine. Added amino acid sequence can be devised to generate an affinity ligand binding site, for convenience and ease of purification. The data reported herein were obtained with a r-HCV-RDRP having an N-terminal MA (Met-Ala) dipeptide, giving an N-terminal sequence MASMSY (SEQ ID NO:6) instead of the predicted SMSY sequence of the most natural isolates of HCV NS5B protein. The coding sequence of NS5B is accordingly modified to include a met codon (ATG) at the 5xe2x80x2-end, as well as, optionally, codons for other amino acids to be included or deleted. Minimal modifications are preferred, in order to avoid potential deleterious effects on enzyme activity, and to avoid creating artificial epitopes. The r-HCV-RDRP can be expressed in procaryotic or eucaryotic cells to yield active RDRP. The expression of active r-HCV-RDRP in E. coli demonstrates that no other HCV-encoded protein is necessary for polymerase activity.
Individual isolates of HCV-RDRP differ widely in their activity, due to differences in amino acid sequence. The present invention introduces the concept of an optimized sequence, whereby specific, directed amino acid substitutions are made, starting from a single original isolate. Individual amino acid substitutions are generated by a series of site specific mutations of the coding region of the original isolate, using known methods. The purpose of the site-specific amino acid substitutions is to enhance catalytic properties of the enzyme. Such properties include, but are not limited to, reaction rate, template specificity, processivity, yield of full length products, ease of purification, and stability, both during purification and during storage, and adaptability to an in vivo assay.
The invention further provides methods for rapid and efficient purification of an r-HCV-RDRP expressed in procaryotic cells, allowing for milligram quantity preparations or r-HCV-RDRP at a purity of at least 95 % as determined by SDS-PAGE (See FIG. 9).
The invention further provides r-HCV-RDRP in solubilized form, and a method of solubilization without destroying activity.
The invention also provides methods for purifying solubilized HCV-RDRP. One such method, to be used in combination with others, is affinity chromatography, using antibody to r-HCV-RDRP as the affinity ligand. Other affinity ligands are obtained by a combinatorial library approach as described, e.g., by Wu, J. et al. (1994) Biochemistry 33:14825-14833; and Ohlmeyer, M. H. J. et al. (1993) Procl. Nat. Acad. Sci. USA 90:10922-10926.
The invention also provides for enzyme sequence modification by adding an affinity tag to enhance ease of purification. The use of an oligo-histidine tag for purification by chromatography on a chelated metal column is described herein.
In addition, the invention provides polyclonal or monoclonal antibodies specific for HCV-RDRP. Such antibodies can be made by known techniques, using the purified enzyme as antigen. Such antibodies bind either r-HCV-RDRP or wild-type HCV-RDRP. The availability of such antibodies makes it possible to prepare an affinity-labeled chromatography matrix for rapid purification of HCV-RDRP. The antibody also makes possible rapid detection of HCV-RDRP in biological materials, for example, in serum of HCV-infected patients.
The invention further provides a method for transfecting a mammalian cell with HCV-RDRP and expressing the enzyme within the cell. Consequently, the invention also provides a transfected mammalian cell line expressing r-HCV-RDRP. Such cells are useful for assaying the effects of candidate anti-viral compounds as inhibitors of RDRP activity. For measuring activity in mammalian cells, the full length enzyme having an intact C-terminal sequence (un-truncated) is considered to be the form most likely to respond to potential inhibitors as the viral enzyme would in infected cells.
Therefore, the invention also provides a method for screening possible inhibitors of RDRP activity in vivo. Compounds with inhibitory activity can have anti-viral activity, since inhibition of the polymerase inhibits viral replication and expression of virus gene products. The in vitro assay is advantageous because it can rule out compounds which cannot enter the infected cell. One class of attractive candidate compounds is the nucleoside analogs; compounds which after being modified (phosphorylated) within cells can bind to substrate sites on the enzyme or which can be incorporated into a newly synthesized RNA but whose presence there disrupts normal function of the HCV polymerase or further replication of an RNA containing the analog. Acyclovir is one example of a very effective and safe nucleoside analogue that inhibits DNA virus replication by inhibiting a viral polymerase (DNA-dependent DNA polymerase) and interfering with primer-template function (chain termination). Such analogs are almost always effective only in the nucleotide triphosphate form. The in vitro assay provides a convenient method of administering the compound in its nucleoside form or nucleoside monophosphate form, allowing endogenous metabolic activity of the cell to convert that form to the active triphosphate, thereby avoiding a step of chemical synthesis of the triphosphate, as would be required for an in vivo assay.
A method for measuring HCV-RDRP activity in vitro is also provided. Such an assay permits identification of the enzyme and evaluation of its concentration during purification. In addition, the assay provides an additional, in vitro, method for screening potential inhibitors of RDRP as candidate anti-viral agents.
In principle, any compound can be tested as a candidate RDRP inhibitor. Certain classes of compounds are considered attractive candidates. These include, without limitation, nucleoside analogs, oligonucleotides and peptides. Certain compounds having planar, polycyclic-aromatic characteristics are also potential inhibitors. It will be understood that compounds identified as effective RDRP inhibitors must be further screened for toxicity, bioavailability, side effects and the like before being tested as therapeutic agents. Nevertheless, the initial identification as an inhibitor of HCV-RDRP is an essential first step in the development of an anti-viral therapy. It will also be recognized that an inhibitor of r-HCV-RDRP will also inhibit wild-type HCV-RDRP.
In another aspect of the invention, the existence of purified HCV-RDRP or r-HCV-RDRP makes it possible to detect and measure antibodies to RDRP present in the serum of an HCV-infected patient. The fact that such antibodies exist at all is in itself a finding made possible by the expression and preparation of purified r-HCV-RDRP according to the invention. The existence of circulating antibodies to HCV-RDRP in infected serum may be due to lysis of infected cells and release of HCV-RDRP into the extracellular fluids and bloodstream, where it can stimulate an antibody response. As the disease fluctuates in severity, the amounts of HCV-RDRP released and the amounts of antibody thereto would also fluctuate. Therefore, the amount of antibody to HCV-RDRP present in a patient""s serum can be used as an indicator, not only of the presence of infection, but of its severity at a given time. The assay for anti-HCV-RDRP can serve as a means of diagnosing infection and also as a means of monitoring the course of the disease over time or in response to treatment. The assay for anti-HCV-RDRP can be carried out by a variety of known techniques, such as the gel separation method described herein. Other suitable methods include ELISA, and radioimmunoassay. A sandwich-type assay, using immobilized r-HCV-RDRP to capture the antibody can then use an anti-immunoglobulin reagent tagged with an appropriate marker such as an enzyme, radioisotope, fluorescent molecule or chemiluminescent marker or the like, all as understood by those skilled in the art. (Antibodies: A laboratory manual, Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988) pp. 553-611.)